Volume 25, Issue 16, Pages 2177-2183 (August 2015) Zeta-Tubulin Is a Member of a Conserved Tubulin Module and Is a Component of the Centriolar Basal Foot in Multiciliated Cells  Erin Turk, Airon A. Wills, Taejoon Kwon, Jakub Sedzinski, John B. Wallingford, Tim Stearns  Current Biology  Volume 25, Issue 16, Pages 2177-2183 (August 2015) DOI: 10.1016/j.cub.2015.06.063 Copyright © 2015 Elsevier Ltd Terms and Conditions

Current Biology 2015 25, 2177-2183DOI: (10.1016/j.cub.2015.06.063) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 1 Zeta-Tubulin Is a Conserved Tubulin that Associates with TRiC/CCT (A) Presence or absence of ZED module tubulins in representative organisms. (B) Unrooted phylogenetic tree, based on Clustal Omega alignment. (C–E) Sucrose gradient sedimentation of zeta-tubulin from (C) A6 cells, (D) eggs, and (E) whole oviduct. Lysates were separated on 10%–40% sucrose gradients and fractions probed for zeta-tubulin, the TRiC/CCT component CCT2, and gamma-tubulin. Molecular weights in kilodaltons (left) and increasing sucrose concentration, left to right (above), are indicated. The dividing line in (E) indicates the joint between two membranes, probed and developed identically. See also Table S1 and Figure S1. Current Biology 2015 25, 2177-2183DOI: (10.1016/j.cub.2015.06.063) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 2 Zeta-Tubulin Localizes to the Basal Foot in Multiciliated Cells (A) Dissociated Xenopus multiciliated oviduct cells stained for zeta-tubulin (green), acetylated tubulin (red), and DAPI (blue). The scale bar represents 5 μm. (B) Transmission EM of oviduct tissue stained with zeta-tubulin antibody and 10-nm gold-conjugated secondary antibody. Arrowheads indicate the basal body, and arrows indicate labeling of the basal foot. The scale bars represent 100 nm. (C) Confocal image of live tadpole epidermal multiciliated cell expressing GFP-zeta-tubulin (green), CLAMP-RFP (red), and centrin-BFP (blue). The arrow shows the direction of ciliary beating. The scale bar represents 5 μm. (D) Confocal image of live tadpole epidermal multiciliated cell expressing EMTB-3XGFP (green), mCherry-zeta-tubulin (red), and centrin-BFP (blue). The arrow shows the direction of ciliary beating. The scale bar represents 5 μm. See also Figures S2 and S3. Current Biology 2015 25, 2177-2183DOI: (10.1016/j.cub.2015.06.063) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 3 Zeta-Tubulin Depletion Disrupts Basal Body Orientation and Spacing (A) Tadpole epidermal multiciliated cells in morphant and control embryos co-expressing CLAMP-GFP (green) and centrin-(RFP or BFP) (red). Clumps of basal bodies are indicated by arrowheads and insets. The scale bars represent 5 μm. (B) Quantification of mean rootlet angle from experiments as in (A). Each arrow represents one cell, where length indicates uniformity of rootlet angles in that cell. Mean cellular rootlet orientation was statistically similar between morphants and controls (mean vector angle −64.4° in controls and −74.8° in morphants; Watson-Williams). The number of cells counted is as in (C). (C) Vector length of plots shown in (B) is significantly reduced in morphants (∗∗∗p < 0.0001; Mann-Whitney). n indicates number of cells counted, with total number of embryos in parentheses. Error bars represent the SEM. (D and E) Quantification of basal body clumping phenotype from experiments as in (A). n indicates the number of cells counted, with total number of embryos in parentheses (shown in E). (D) The distance between each basal body and its nearest neighbor is shown as average percentage of binned nearest neighbor distances for each condition. (E) Mean distance between basal bodies is shown; this is significantly reduced in morphants (∗∗∗p < 0.0001; Mann-Whitney). In (E), error bars represent the SEM. See also Figure S4. Current Biology 2015 25, 2177-2183DOI: (10.1016/j.cub.2015.06.063) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 4 Zeta-Tubulin Function and ZED Tubulin Localization (A) Microtubule organization in tadpole epidermal multiciliated cells visualized by co-expressing EMTB-3XGFP (green) and centrin-RFP (red). The scale bars represent 5 μm. (B) Tadpole epidermal multiciliated cells expressing centrin-BFP (white) fixed and stained with Alexa Fluor 568 phalloidin (red). Apical actin (apical inset) and subapical foci (subapical slice; ∼1.2 μm below the apical section) are shown. The scale bars represent 5 μm. (C) Tadpole epidermal multiciliated cell expressing GFP-epsilon-tubulin (green), mCherry-zeta-tubulin (red), and centrin-BFP (blue). The scale bar represents 5 μm. (D) Mouse tracheal epithelial cell cultures infected with GFP-zeta-tubulin-expressing lentivirus and stained for GFP (green), centrin (red), and DAPI (blue). x-z projections (below) show a slice through the area marked by the white rectangle. The scale bar represents 5 μm. See also Figures S3 and S4. Current Biology 2015 25, 2177-2183DOI: (10.1016/j.cub.2015.06.063) Copyright © 2015 Elsevier Ltd Terms and Conditions